This invention relates to methods and compositions useful to prevent and treat Clostridium difficile-associated diseases.
Clostridium difficile-associated diseases (CDAD) are the most frequently identified causes of nosocomial diarrhea (that is, in healthcare facilities), producing both endemic and epidemic diarrhea. CDAD incidence ranges from  less than 1% to 7.8% of hospital discharges. CDAD prolongs hospitalization, increases the costs of care, and causes considerable morbidity, especially in the 10-20% who relapse, and a mortality of 0.6-2.3%. Elderly patients and those who have long hospital stays are at particularly high risk.
Horizontal transmission of C. difficile in the hospital to susceptible patients receiving antibiotics accounts for the vast majority of CDAD incidence. No infection control method has been widely successful at preventing transmission, and prophylactic measures aimed at preventing symptoms if transmission occurs have proven cumbersome, ineffective, or both. Therefore, new and innovative approaches to prevent CDAD are needed.
In animals, C. difficile-related diseases cause great economic losses. Notably, in horse breeding, young foals are extremely susceptible, infection generally resulting in death; chinchillas are also vulnerable. Antibiotic treatment in animals is not only expensive, but is not completely effective.
There is a need for relatively inexpensive and effective prophylactic methods and treatments.
Although use of a non-toxigenic strain of C. difficile has been suggested as a solution, selection of strains has been random, and results ambiguous or discouraging. For example, Wilson and Sheagren (1983) report that colonization of cefotoxin-treated hamsters with a strain of C. difficile, said to be non-toxigenic, but not further characterized, before challenge with toxigenic C. difficile resulted in only 72% improvement in survival compared to controls. Even more discouraging, when toxigenic and non-toxigenic strains were given at the same time, no significant survival benefit was obtained.
Borriello and Barclay (1985) reported that prior colonization of clindamycin-treated hamsters with non-toxigenic strains of C. difficile protected them for at least a short time period from subsequent colonization with a toxigenic strain. The non-toxigenic strains were given designations, but were not further characterized. In total, 13 of 18 xe2x80x9cprotectedxe2x80x9d hamsters survived for up to 27 days, whereas all 27 animals challenged with the toxigenic strain alone died within 48 hours. However, even in toe xe2x80x9cprotectedxe2x80x9d animals, the toxigenic strain eventually became dominant and caused disease, and death occurred in most cases due to infection. The authors concluded that the extent to which this sort of approach may be therapeutically useful was difficult to assess.
Seal et al. (1987) reported that two patients (designated A and B) with relapsing C. difficile diarrhea following metronidazole and vancomycin therapy were colonized with a non-toxigenic avirulent C. difficile strain given orally in three doses. Patient A did not suffer a further relapse during the four months that she was studied after treatment with non-toxigenic C. difficile. Patient B did suffer a relapse, but it was reported as milder than previous relapses. The authors acknowledged that there was no showing that the bacteriotherapy actually prevented or mitigated relapse. Indeed, in one of the two patients, it had not prevented relapse. Obviously, conclusions on only two patients are questionable. The authors also stated that there was a need for further studies before this approach should be considered for trials in the hospital.
Corthier and Muller (1988) reported that administering a non-toxigenic strain of C. difficile to gnotobiotic mice from 18 hours to 10 days before challenge with toxigenic C. difficile resulted in 100% survival, however, the controls exhibited 60% survival in this experiment, suggesting only about 40% improvement. It cannot be determined from the article how long this level of protection persisted. The mice appear to have been observed for only eight days after administration of the toxigenic C. difficile. 
Borriello (1988) reviewed bacteriotherapy approaches for the prevention and treatment of C. difficile infection of the gut, including use of whole fecal mixtures, synthetic mixtures of fecal organisms, lactobacilli, the yeast Saccharomyces boulardii, and non-toxigenic C. difficile in vitro, and in vivo in animals and in vivo in humans. He concluded that all of the bacteriotherapy approaches need to be further evaluated, but that a non-toxigenic strain of C. difficile may fulfill criteria for a xe2x80x9ccleanerxe2x80x9d and well defined preparation.
No reports of attempts to use C. ditficile bacteriotherapy to protect against CDAD are known since 1988. Experimental bacteriotherapy is in clinical use in the U.S. with Saccharomyces boulardii, a yeast, which is reported to show some benefit in reducing relapses and in preventing antibiotic-associated diarrhea, but is not effective against C. difficile diarrhea (McFarland et al., 1994; Surawicz et al., 1989). A problem with Saccharomyces is that twice daily administration is required for four weeks, making compliance difficult for patients.
The successful use of rectal instillation of feces and bacterial mixtures of fecal organisms to treat chronic relapsing C. difficile has been reported in six patients (Tvede and Rask-Madsen, 1989). Use of bacteriotherapy has not been wide-spread due to reluctance by physicians and patients aesthetically to use fecal preparations. At present, methods and compositions for specific prevention of C. difficile diarrhea have not been reported.
The present invention provides methods and compositions for preventing and treating CDAD (C. difficile-associated disease) in subjects including humans and non-human animals, e.g. mammals and birds. xe2x80x9cSubjectsxe2x80x9d are persons or animals who have received antimicrobials o antineoplastics (which also have antimicrobial activity). For prevention of C. difficile disease, a composition of a selected non-toxigenic strain of C. difficile is administered within about 24 hrs. after antibiotic (antimicrobial) or antineoplastic agents. This is to allow time for suppression of the normal intestinal flora, but not enough time for toxigenic strains of C. difficile to become established in the gastrointestinal tract. Examples of types of antimicrobial agents known to cause risk of C. difficile disease in human and non-human animals or birds include cephalosporins, clindamycin, ampicillin, tetracyclines.
For treatment of subjects who have C. difficile disease, first antibiotics (usually vancomycin or metronidazole) must be administered that are directed to control of the disease, thenxe2x80x94to prevent a relapse, the compositions of the present invention are administered.
The methods of the present invention comprise administering to the subject an effective amount of spores (generally in the order of 5xc3x97105 to 106 colony-forming units) of a selected non-toxigenic strain of C. difficile. Higher or repeated doses may be required for treatment than for prevention. xe2x80x9cEffectivenessxe2x80x9d is determined by clinical criteria showing the risk of developing CDAD is reduced by 80% or more compared to comparably exposed patients or animals in the same environment.
An aspect of the invention is compositions for use in the method, said compositions including selected non-toxigenic strains of C. difficile. Certain non-toxigenic strains (types) of C. difficile are found to prevent disease better against specific toxigenic types of C. difficile than against other toxigenic types. Methods to select these strains are an aspect of the present invention.
A suitable non-toxigenic strain is a strain selected from the M, T, C, AP or other non-toxigenic groups as described by Clabots et al. (1993) or as genetically engineered to be non-toxigenic. These groups are selected because their relative high frequencies as isolates from persons or humans in which colonization has occurred, predicts their success as colonizers for purposes of the invention. Generally, relatively frequent groups occur in at least 5% of the non-toxigenic isolates, preferably in at least 15%; and more preferably in at least 25% of the isolates. Similarly, within a group, preferred strains are those that are most frequent. A single purified strain of the M group, or of another single group, or a combination of strains within a group or a combination of strains from different groups, are suitable. M group strains, in particular M3 and M23 are preferred singly or in combination with other strains. Also provided are pharmaceutical compositions and unit dosage forms comprising a strain selected from the non-toxigenic C. difficile groups, or a combination of strains.
In some conditions, it is advantageous to combine two or more non-toxigenic strains that have complementary spectrums of prevention in order to achieve prevention of disease against a broader range of toxigenic C. difficile organisms. This strategy is based on the assumption that the two or more strains each colonize effectively when administered together and at the same time (so that neither strain has the advantage of being the first to arrive in the GI tract). Equivalent amounts of each type in the combination are a starting point for administration to prevent any one type from having an advantage in numbers. Ratios are adjusted if initial amounts are not effective.
It is possible to genetically engineer non-toxigenic strains of C. difficile by removing or manipulating the two genes responsible for production of the C. difficile toxins, A and B, so as to create strains that no longer produce the toxins responsible for causing C. difficile-associated disease (CDAD). Such strains are suitable for the practice of the present invention to prevent CDAD in the same manner as naturally non-toxigenic strains are disclosed herein. Such strains would have to undergo extensive safety and efficacy trials in animals and humans to assure that they were safe, e.g. had not acquired any inadvertent virulence properties as a result of genetic engineering. They would also have to be shown to be efficacious as a preventive measure by the methods disclosed herein, and to not revert back to their original toxigenic state or reacquire the toxin genes.
The methods and compositions of the present invention are useful to prevent and treat disease in humans or non-human animals, and are particularly useful for preventing and treating multiple relapses.